We have examined the functional role of some active site residues of E. coli aspartate aminotransferase by using kinetic analysis on mutant enzymes produced by the site-directed mutagenesis. Aspartate aminotransferase has been the most extensively studied representative of many transaminases ; X-ray analysis has been done. Our findings are as follows :1. Lys258 is essential for catalysis, acting as a catalytic base to withdraw an a -proton from the amino acid substrate, which is a prerequisite for the catalysis.2. A negative charge at position 222, and a hydrogen bond between the hydroxyl group of Tyr225 and the unprotonated hydroxyl group of the coenzyme probably help in lowering the electron density of the coenzyme to facilitate the a- proton removal.3. Arg292 and Arg386 are essential for the recognition of the dicarboxylic substrates.4. Substitution of Arg292 to uncharged residues greatly enhanced the catalytic efficiency of the transamination of neutral amino acid without. any effect on the binding.5. The endole ring of Trpl4O not only regulates the rotational movement of the coenzyme ring during the catalysis, but it may be also involved in the binding of the carboxyl side chain of the dicarboxylic acid substrates.6. The phenol group of Tyr7O is essential for the stabilization of the transition states with all substrates. The presence of the benzen ring at position 70 is necessary to recognize the glutanate-2-oxoglutarate substrate pair.